The staphylococci are Gram-positive spherical cells, usually arranged in grape-like irregular clusters. Some are members of the normal flora of the skin and mucous membranes of humans, others cause suppuration, abscess formation, a variety of pyogenic infections, and even fatal septicemia. Pathogenic staphylococci often hemolyze blood, coagulate plasma, and produce a variety of extracellular enzymes and toxins. The most common type of food poisoning is caused by a heat-stable staphylococci enterotoxin.
The genus Staphylococcus has at least 30 species. Three main species of clinical importance are Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus haemolyticus. Staphylococcus aureus is coagulase-positive, which differentiates it from the other species. S. aureus is a major pathogen for humans. Almost every person has some type of S. aureus infection during a lifetime, ranging in severity from food poisoning or minor skin infections to severe life-threatening infections. The coagulase-negative staphylococci are normal human flora which sometimes cause infection, often associated with implanted devices, especially in very young, old and immunocompromised patients. Approximately 75% of the infections caused by coagulase-negative staphylococci are due to parasitic S. epidermidis. Infections due to Staphylococcus haemolyticus, Staphylococcus hominis, and other species are less common. S. saprophyticus is a relatively common cause of urinary tract infections in young women.
Staphylococcus bacteria such as S. aureus thus cause a spectrum of infections that range from cutaneous lesions such as wound infections, impetigo, and furuncles to life-threatening conditions that include pneumonia, septic arthritis, sepsis, endocarditis, and biomaterial related infections. S. aureus colonization of the articular cartilage, of which collagen is a major component, within the joint space appears to be an important factor contributing to the development of septic arthritis. Hematogenously acquired bacterial arthritis remains a serious medical problem. This rapidly progressive and highly destructive joint disease is difficult to eradicate. Typically less than 50% of the infected patients failing to recover without serious joint damage. S. aureus is the predominant pathogen isolated from adult patients with hematogenous and secondary osteomyelitis.
In hospitalized patients, Staphylococcus aureus is a major cause of infection. Initial localized infections of wounds or indwelling medical devices can lead to more serious invasive infections such as septicemia, osteomyelitis, mastitis and endocarditis. In infections associated with medical devices, plastic and metal surfaces become coated with host plasma and matrix proteins such as fibrinogen and fibronectin shortly after implantation. The ability of Staphylococcus bacteria such as S. aureus to adhere to these proteins is essential to the initiation of infection. Vascular grafts, intravenous catheters, artificial heart valves, and cardiac assist devices are thrombogenic and prone to bacterial colonization. S. aureus is the most damaging pathogen of such infections, and other Staphylococci bacteria such as S. epidermidis are also responsible for a significant amount of dangerous infections, particularly those associated with implanted devices.
There is a strong and rapidly growing need for therapeutics to treat infections from Staphylococcus bacteria such as S. aureus and S. epidermidis infections which are effective against antibiotic resistant strains of the bacteria. The U.S. National Institutes for Health has recently indicated that this goal is now a national priority.
The successful colonization of the host is a process required for most microorganisms to cause infections in animals and humans. Microbial adhesion is the first crucial step in a series of events that can eventually lead to disease. Pathogenic microorganisms colonize the host by attaching to host tissues or serum conditioned implanted biomaterials, such as catheters, artificial joints, and vascular grafts, through specific adhesins present on the surface of the bacteria. MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) are a family of cell surface adhesins that recognize and specifically bind to distinct components in the host""s extracellular matrix. Once the bacteria have successfully adhered to and colonized host tissues, their physiology is dramatically altered and damaging components such as toxins and proteolytic enzymes are secreted. Moreover, adherent bacteria often produce a biofilm and quickly become more resistant to the killing effect of most antibiotics.
For example, S. aureus is known to express a repertoire of different MSCRAMMs that can act individually or in concert to facilitate microbial adhesion to specific host tissue components. MSCRAMMs provide an excellent target for immunological attack by antibodies. The presence of the appropriate anti-MSCRAMM high affinity antibodies has a double-edged attack, first the antibodies prevent microbial adherence and second the increased titers of MSCRAMM antibodies facilitate a rapid clearance of the organism from the body through bacterial lysis, opsonization, phagocytosis and complement activation.
Immunoglobulins (A, D, E, G, and M) are used by the body as a primary defense to infections. Complement, available as a precursor protein which is activated by the presence of microorganisms and globulins, also exhibits antibacterial activities. After previous antigenic exposure, the immune system produces a series of globulins which attach to and coat bacteria or neutralize viruses so that they are readily recognized, phagocytized and destroyed by neutrophils and macrophages. Foreign proteins of invading organisms also stimulate a humoral immune response which over a period of time from three to six weeks amplifies the number of cells designed to recognize and destroy specific invaders.
In the last decade, intravenous immunoglobulin (IVIG) therapy has become a major treatment regime for bacterial infections, especially in immunocompromised patients (Siber, New Eng. J. Med., 327:269-271, 1992). IVIG therapy has exhibited efficacy against more than thirty-five diseases caused by immunopathologic mechanisms. Passive immunization against infections has been particularly successful with immune globulins specific for tetanus, hepatitis B, rabies, chicken pox and cytomegalovirus. There has been an inconsistent and disappointing response to the use of immunoglobulins to prevent nosocomial infections, likely due to the variety of strains of bacteria found in hospitals and the emergence of new serotypes. Passive immunization requires the presence of high and consistent titers of antibodies to the infecting pathogens.
Supplemental immunoglobulin therapy has been shown to provide some measure of protection against certain encapsulated bacteria such as Hemophilus influenzae and Streptococcus pneumoniae. Infants who are deficient in antibody are susceptible to infections from these bacteria and bacteremia and sepsis are common. When anti-Streptococcal and anti-Hemophilus antibodies are present, they provide protection by promoting clearance of the respective bacteria from the blood. In the case of antibody to Staphylococcus, the potential use of supplemental immunoglobulin to prevent or treat infection has been much less clear.
Early attempts to treat Staphylococcus infections focused on the potential use of supplemental immunoglobulin to boost peritoneal defenses, such as opsonic activity, in patients receiving continuous ambulatory peritoneal dialysis. Standard intravenous immunoglobulin (IVIG) was shown to have lot to lot variability for opsonic activity to S. epidermidis (L. A. Clark and C. S. F. Easmon, J. Clin. Pathol. 39:856 (1986)). In this study, one third of the IVIG lots tested had poor opsonization with complement, and only two of fourteen were opsonic without complement. Thus, despite the fact that the IVIG lots were made from large plasma donor pools, good opsonic antibody to S. epidermidis was not uniformly present. Moreover, this study did not examine whether IVIG could be used to prevent or treat S. epidermidis infections or bacterial sepsis.
Prior studies have associated coagulase-negative Staphylococcus bacteria, such as S. epidermidis, as the most common species causing bacteremia in neonates receiving lipid emulsion infusion (Freeman, J. et al., N. Engl. J. Med. 323:301, 1990). These neonates had low levels of opsonic antibody to S. epidermidis despite the fact that the sera had clearly detectable levels of IgG antibodies to S. epidermidis peptidoglycan (Fleer, A. et al., J. Infect. Dis. 2:426, 1985). This was surprising because anti-peptidoglycan antibodies were presumed to be the principal opsonic antibodies. Thus, while suggesting that neonatal susceptibility to S. epidermidis might be related to impaired opsonic activity, these studies also suggested that many antibodies directed against S. epidermidis are not opsonic and would not be capable of providing protection when given passively to neonates.
In addition, an antigen binding assay was used to analyze IgG antibody to S. epidermidis in patients with uncomplicated bacteremia and those with bacteremia and endocarditis (F. Espersen et al., Arch. Intern. Med. 147:689 (1987)). This assay used an ultrasonic extract of S. epidermidis to identify S. epidermidis specific IgG. None of the patients with uncomplicated bacteremia had IgG antibodies to S. epidermidis. These data suggest that IgG does not provide effective eradication of S. epidermidis from the blood. In addition, 89% of bacteremic patients with endocarditis developed high levels of IgG to S. epidermidis. In these patients, IgG was not protective since high levels of IgG antibody were associated with serious bacteremia and endocarditis. Based on these studies, the protective role of IgG in S. epidermidis sepsis and endocarditis was questionable, especially in the presence of immaturity, debilitation, intralipid infusion, or immunosuppression.
Animal studies in the literature that demonstrated immunoglobulin protection against Staphylococcus infections have shown strain specificity by enzyme-linked immunosorbent assays (ELISA) and have utilized normal adult mice in protection studies. Animal models typically have used mature animals with normal immunity with unusually virulent strains or overwhelming-challenge doses of bacteria. Human patients are generally immunologically immature or debilitated. Human patients also get somewhat indolent infections with low virulence pathogens such as S. epidermidis with death usually attributable to secondary complications. Models that have used unusual strains or overwhelming bacterial doses, generally induce rapid fulminant death. These are important factors since antibodies generally work in concert with the host cellular immune system (neutrophils, monocytes, macrophages and fixed reticuloendothelial system). The effectiveness of antibody therapy may therefore be dependent on the functional immunologic capabilities of the host. To be predictive, animal models must closely emulate the clinical condition in which the infection would occur and capture the setting for therapy. Moreover, the animal studies have yielded inconsistent results.
One model has been reported which used an unusually virulent strain of S. epidermidis. Infected-mature mice developed 90 to 100% mortality within 24 to 48 hours (K. Yoshida et al., Japan. J. Microbiol. 20:209 (1976)). Antibody to S. epidermidis surface polysaccharide was protective in these mice. Protection was shown to occur with an IgM fraction, but not the IgG fraction (K. Yoshida and Y. Ichiman, J. Med. Microbiol. 11:371 (1977)). This model, however, presents a pathology which is very different from that seen in typically infected patients. Intraperitoneally-challenged mice developed symptoms of sepsis within minutes of receiving the injection and died in 24 to 48 hours. This particular pathology is not observed in Staphylococcus infected humans. The highly virulent strain of S. epidermidis may represent an atypical type of infection, moreover, isolates of S. epidermidis from infected humans did not kill mice in this model.
In 1987, these animal studies were extended to include the evaluation of antibodies in human serum against selected virulent strains of S. epidermidis (Y. Ichiman et al., J. Appl. Bacteriol. 63:165 (1987)). In contrast to the previous data, protective antibody was found in the IgA, IgM and IgG immunoglobulin fractions. A definitive role for any single class of immunoglobulin (IgG, IgM, IgA) could not be established.
In this animal model, normal adult mice were used and mortality was determined. Death was considered to be related to the effect of specific bacterial toxins, not sepsis (K. Yoshida et al., Japan J. Microbiol. 20:209 (1976)). Most clinical isolates did not cause lethal infections, and quantitative blood cultures were not done. Moreover, this study provided little insight as to whether antibody could successfully prevent or treat S. epidermidis sepsis in immature or immunosuppressed patients.
In a later study, serotype specific antibodies directed against S. epidermidis capsular polysaccharides were tested in the animal model. Results showed that serotype-specific antibodies were protective, but that each antibody was directed against one serotype as measured by ELISA. Protection was equally serotype specific. Protection against heterologous strains did not occur. In addition, it was concluded that protection was afforded by the IgM antibody.
There has been no compelling evidence that IVIG would be effective to treat S. epidermidis infections or sepsis, particularly where the patients are immature or immune suppressed or where multiple S. epidermidis serotypes are involved. Thus, for example, a recent and extensive review of the pathogenesis, diagnosis, and treatment of S. epidermidis infections does not include immunoglobulin as a potential prophylactic or therapeutic agent (C. C. Patrick, J. Pediar. 116:497 (1990)). In addition, there have been no U.S. patents which describe the effective use of IVIG therapy in conjunction with antibodies to MSCRAMMs such as described above.
U.S. Pat. No. 5,505,945 discloses compositions for passive immunity that contain a full repertoire of immunoglobulins, including IgA, IgM, and IgG to combat infections from microorganisms and viruses at wound, surgical, or burn sites. The compositions contain elevated antibody titers for several pathogens, including S. aureus, Coagulase Negative Staphylococci Enterococci, S. epidermidis, P. aeruginose, E. coli, and Enterobacter spp. However, these compositions are specifically designed to avoid the use of intravenous immunoglobulin or IVIG therapy, and instead are applied in the form of ointments, creams, sprays and the like which are designed for topical application only.
U.S. Pat. No. 4,717,766 discloses a method of preparing high titer anti-respiratory syncytial virus intravenous immunoglobulins.
U.S. Pat. No. 5,219,578 describes a composition and method for immunostimulation in mammals, and specifically describes the isolation of an IgG fraction from goats free from foreign or artificially induced antigens and the utilization of the isolated immunoglobulins fraction to induce a stimulated immune response.
U.S. Pat. No. 5,548,066 describes a method for drawing blood from a donor animal, permitting blood to clot, separating liquid from cellular material, and then clarifying, concentrating and sterilizing the product.
U.S. Pat. No. 4,412,990 discloses an intravenous pharmaceutical composition containing immunoglobulin (IgG) and fibronectin that exhibits a synergistic opsonic activity which results in enhanced phagocytosis of bacteria, immune complexes and viruses.
U.S. Pat. No. 4,994,269 discloses the topical use of monoclonal antibodies for the prevention and treatment of experimental P. aeruginosa lung infections. Specifically, the antibodies are administered via aerosol spray to the lungs. Results show beneficial effects in the treatment of affected patients.
U.S. Pat. No. 4,714,612 discloses the use of a non-specific gamma globulin IgG in a mouthwash for the prevention of gingivitis. Another mouthwash with monoclonal antibodies is described by Ma et al. in Arch. Oral Biol., 35 Supp: 115S-122S, in 1990. The monoclonal antibodies were specific for Streptococcus mutans, and patients treated with the mouthwash remained free of S. mutans for up to two years. Those who did not take the mouthwash experiences recolonization of S. mutans within two days.
U.S. Pat. Nos. 5,718,889 and 5,505,945 describe the direct, concentrated local delivery of passive immunity which is accomplished by applying a composition having a full repertoire of immunoglobulins (IgG, IgM and IgA) to biomaterials, implants, tissues, and wound and burn sites.
U.S. Pat. No. 5,571,511 describes the use of immunoglobulin from individual samples or pools of serum, plasma, whole blood, or tissue for the treatment of a Staphylococcus infection. Immunoglobulin is identified by performing a first assay to identify immunoglobulin which is reactive with a preparation of a first Staphylococcus organism, performing a second assay to identify immunoglobulin which is reactive with a preparation of a second Staphylococcus organism, and selecting immunoglobulin which is reactive with the preparations from both the first and second Staphylococcus organisms. Reactivity is determined in immunological assays which may be binding assays, opsonization assays, or clearance assays. Preferably, the preparations of the first and the second Staphylococcus organisms are derived from different serotypes or different species, such as S. epidermidis and S. aureus, and more preferably, the first preparation is from S. epidermidis (Hay, ATCC 55133).
U.S. Pat. No. 5,412,077 describes the screening of plasma samples for effective antibody titers for the treatment or prophylaxis of an infection caused by a respiratory virus.
Accordingly, there still remains a need to provide more effective products and methods which make use of antibodies against MSCRAMMs and can be utilized in methods of intravenous immunoglobulin therapy so as to prevent and/or treat Staphylococcus infections, and preferably those that can exhibit a broad spectrum immunization against various strains of Staphylococcus bacteria.
Historically, studies on bacterial adherence have focused primarily on Gram-negative bacteria, which express a wide variety of adhesive proteins on their cell surface (Falkow, S., et al.; Cell, 65:1099-1102, 1992). These adhesins recognize specific glycoconjugates exposed on the surface of host cells (particularly epithelial layers). Employing the lectin-like structures in attachment allows the microorganism to efficiently colonize the epithelial surfaces. This provides the bacteria an excellent location for replication and also the opportunity to disseminate to neighboring host tissues. It has been demonstrated that immunization with pilus adhesins can elicit protection against microbial challenge, such as in Hemophilus influenza induced otitis media in a chinchilla model (Sirakova et al., Infect. Immun, 62(5):2002-2020, 1994), Moraxella bovis in experimentally induced infectious bovine keratoconjunctivitis (Lepper et al., Vet Microbiol, 45(2-3):129-138, 1995), and E. coli induced diarrhea in rabbits (McQueen et al., Vaccine, 11:201-206, 1993). In most cases, immunization with adhesins leads to the production of immune antibodies that prevent infection by inhibiting bacterial attachment and colonization, as well as enhancing bacterial opsonophagocytosis and antibody-dependent complement-mediated killing.
The use of molecules that mediate the adhesion of pathogenic microbes to host tissue components as vaccine components is emerging as a critical step in the development of future vaccines. Because bacterial adherence is the critical first step in the development of most infections, it is an attractive target for the development of novel vaccines. An increased understanding of the interactions between MSCRAMMs and host tissue components at the molecular level coupled with new techniques in recombinant DNA technology have laid the foundation for a new generation of subunit vaccines. Entire or specific domains of MSCRAMMs, either in their native or site-specifically altered forms, can now be produced. Moreover, the ability to mix and match MSCRAMMs from different microorganisms creates the possibility of designing a single vaccine that will protect against multiple bacteria.
The recent clinical trials with a new subunit vaccine against whooping cough, consisting of the purified Bordatella pertussis MSCRAMMs filamentous hemagglutinin and pertactin, in addition to an inactivated pertussis toxin, are a prime example of the success of this type of approach. Several versions of the new acellular vaccine were shown to be safe and more efficacious than the old vaccine that contained whole bacterial cells (Greco et al., N Eng J Med, 334:341-348, 1996; Gustaffson et al., N Eng J Med, 334:349-355, 1996).
Natural immunity to Staphylococcus infections remains poorly understood. Typically, healthy humans and animals exhibit a high degree of innate resistance to Staphylococcus bacteria such as S. aureus. Protection is attributed to intact epithelial and mucosal barriers and normal cellular and humoral responses. Titers of antibodies to S. aureus components are elevated after severe infections (Ryding et al., J. Med Microbiol, 43(5):328-334, 1995), however to date there is no serological evidence of a correlation between antibody titers and human immunity.
Over the past several decades live, heat-killed, and formalin fixed preparations of S. aureus cells have been tested as vaccines to prevent staphylococcal infections. A multicenter clinical trial was designed to study the effects of a commercial vaccine, consisting of a staphylococcus toxoid and whole killed staphylococci, on the incidence of peritonitis, exit site infection, and S. aureus nasal carriage among continuous peritoneal dialysis patients (Poole-Warren, L. A., et al., Clin Nephrol, 35:198-206, 1991). Although immunization with the vaccine elicited an increase in the level of specific antibodies to S. aureus, the incidence of peritonitis was unaffected. Similarly, immunization of rabbits with whole cells of S. aureus could not prevent or modify any stage in the development of experimental endocarditis, reduce the incidence of renal abscess, or lower the bacterial load in infected kidneys (Greenberg, D. P., et al., Infect Immun, 55:3030-3034, 1987).
Currently there is no FDA approved vaccine for the prevention of S. aureus infections. However, a S. aureus vaccine (StaphVAX), based on the capsular polysaccharide, is currently being developed by NABI (North American Biologicals Inc.). This vaccine consists of type 5 or type 8 capsular polysaccharides conjugated to Pseudomonas aeruginosa exotoxin A (rEPA). The vaccine is designed to induce type-specific opsonic antibodies and enhance opsonophagocytosis (Karakawa, W. W., et al., Infect Immun, 56:1090-1095, 1988). Using a refined lethal challenge mouse model (Fattom, A., et al., Infect Immun, 61:1023-1032, 1996) it has been shown that intraperitoneal infusion of type 5 specific IgG reduces the mortality of mice inoculated intraperitoneally with S. aureus. The type 5 capsular polysaccharide-rEPA vaccine has also been used to vaccinate seventeen patients with end-stage renal disease (Welch, et al., J Amer Soc Nephrol, 7(2):247-253, 1996). Geometric mean (GM) IgG antibody levels to the type 5 conjugate increased between 13 and 17-fold after the first immunization, however no additional increases could be detected after additional injections. Moreover, these vaccination regimens were not able to treat a variety of bacterial strains.
Interestingly, the GM IgM levels of the vaccinated patients were significantly lower than control individuals. Supported by the animal studies, the vaccine has recently completed a Phase II trial in continuous ambulatory peritoneal dialysis patients. The clinical trial showed the vaccine to be safe but ineffective in preventing staphylococcal infections (NABI SEC FORM 10-K405, Dec. 31, 1995). Two possible explanations for the inability of StaphVAX to prevent infections related to peritoneal dialysis in vaccinated patients are that the immunogenicity of the vaccine was too low due to suboptimal vaccine dosing or that antibodies in the bloodstream are unable to affect infection in certain anatomic areas, such as the peritoneum.
Incidence of gram-positive bacteria related sepsis is increasing. In fact between one-third and one-half of all cases of sepsis are caused by gram-positive bacteria, particularly S. aureus and S. epidermidis. In the United States, it can be estimated that over 200,000 patients will develop gram-positive related sepsis this year. Using a mouse model (Bremell, et al., Infect Immun, 59(8):2615-2623, 1991), it has been clearly demonstrated in PCT WO 97/43314 that active immunization with M55 domain of the Col-binding MSCRAMM can protect mice against sepsis induced death. Mice were immunized subcutaneously with either M55 or a control antigen (bovine serum albumin) and then challenged intravenously with S. aureus. Eighty-three percent (35/42) of the mice immunized with M55 survived compared to only 27% of the BSA immunized mice (12/45). This a compilation of three separate studies.
Schennings et al. demonstrated that immunization with fibronectin binding protein from S. aureus protects against experimental endocarditis in rats (Micro Pathog, 15:227-236, 1993). Rats were immunized with a fusion protein (gal-FnBP) encompassing beta-galactosidase and the domains of fibronectin binding protein from S. aureus responsible for binding to fibronectin. Antibodies against gal-FnBP were shown to block the binding of S. aureus to immobilized fibronectin in vitro. Endocarditis in immunized and non-immunized control rats was induced by catheterization via the right carotid artery, resulting in damaged aortic heart valves which became covered by fibrinogen and fibronectin. The catheterized rats were then infected intravenously with 1xc3x9710 5 cells of S. aureus. The number of bacteria associated with aortic valves was determined 11/2 days after the challenge infection and a significant difference in bacterial numbers between immunized and non-immunized groups was then observed.
A mouse mastitis model was used by Mamo, et al., in 1994 (Vaccine, 12:988-992) to study the effect of vaccination with fibrinogen binding proteins (especially FnBP-A) and collagen binding protein from S. aureus against challenge infection with S. aureus. The mice vaccinated with fibrinogen binding proteins showed reduced rates of mastitis compared with controls. Gross examination of challenged mammary glands of mice showed that the glands of mice immunized with fibrinogen binding proteins developed mild intramammary infection or had no pathological changes compared with glands from control mice. A significantly reduced number of bacteria could be recovered in the glands from mice immunized with fibrinogen binding proteins as compared with controls. Mamo then found that vaccination with FnBP-A combined with staphylococcal alpha toxoid did not improve the protection (Mamo, et al., Vaccine, 12:988-992, 1994). Next, Mamo, et al., immunized mice with only collagen binding protein, which did not induce protection against the challenge infection with S. aureus. 
Whole killed staphylococci were included in a vaccine study in humans undergoing peritoneal dialysis (Poole-Warren, et al., Clinical Nephrology 35:198-206, 1991). In this clinical trial, a commercially available vaccine of alpha-hemolysin toxoid combined with a suspension of whole killed bacteria) was administered intramuscularly ten times over 12 months, with control patients receiving saline injections. Vaccination elicited significant increases in the levels of antibodies to S. aureus cells in the peritoneal fluid and to alpha-hemolysin in the serum. However, immunization did not reduce the incidences of peritonitis, catheter-related infections or nasal colonization among vaccine recipients. The lack of protective efficacy in this trial was attributed to a suboptimal vaccine formulation.
Secreted proteins have been explored as components of subcellular vaccines. The alpha toxin is among the most potent staphylococcal exotoxins; it has cytolytic activity, induces tissue necrosis and kills laboratory animals. Immunization with formaldehyde-detoxified alpha toxin does not protect animals from systemic or localized infections, although it may reduce the clinical severity of the infections (Ekstedt, R. D., The Staphylococci, 385-418, 1972).
One study has evaluated the protective efficacy of antibodies to the S. aureus microcapsule in an experimental model of staphylococcal infection (Nemeth, J. and Lee, J. C., Infect. Immun., 61:1023-1032, 1993). Rats were actively immunized with killed, microencapsulated bacteria or passively immunized with high-titer rabbit antiserum specific for the capsular polysaccharide. Control animals were injected with saline or passively immunized with normal rabbit serum. Protection against catheter-induced endocarditis resulting from intravenous challenge with the same strain was then evaluated. Despite having elevated levels of anticapsular antibodies, the immunized animals were susceptible to staphylococcal endocarditis and immunized and control animals had similar numbers of bacteria in the blood.
As described in the Detailed Description of the Invention hereinbelow, a number of patents and published patent applications describe the gene sequences for fibronectin, fibrinogen, collagen, elastin, and MHC II antigen type binding proteins. These patents and patent applications are incorporated by reference in their entirety. These documents teach that the proteins, fragments, or antibodies immunoreactive with those proteins or fragments can be used in vaccinations for the treatment of S. aureus infections. PCT/US97/087210 discloses the vaccination of mice with a combination of a collagen binding protein (M55 fragment), a fibronectin binding peptide (formalin treated FnBPA (D1-D3)) and a fibrinogen binding peptide (ClfA).
Despite the advances in the art of compositions for the treatment of infections from Staphylococcus bacteria such as S. aureus, there remains a need to provide a more effective product, and preferably one that exhibits a broad spectrum immunization against a variety of Staphylococcus bacterial strains. As described in the Detailed Description of the Invention, one approach to generating a prophylactic immunotherapeutic against bacteria is to stimulate donors with a vaccine containing a combination of MSCRAMMs. This approach of generating hyperimmune globulins can create a steady supply of plasma with high levels of the specific types of disease fighting antibodies. MSCRAMM hyperimmune globulins can be used to provide passive immunity against infection in neonates, trauma patients, immunocompromised patients or patients who are immediately at risk and do not have time to mount their own antibody response. Hyperimmune globulins have a high benefit-to-cost ratio, can be produced from a nonhuman or human source and have a high level of physician acceptance based on past usage.
Therefore, it is an object of the invention to provide new therapeutic compositions for active and passive immunization against Staphylococcus infections.
It is another object of the present invention to provide active and passive immunization against mastitis, arthritis, endocarditis, septicemia, osteomyelitis, furunculosis, cellulitis, pyemia, pneumonia, pyoderma, suppuration of wounds, food poisoning, bladder infections and other infectious diseases.
It is another object of the present invention to provide a therapeutic composition that immunizes against Staphylococcus bacteria such as S. aureus and S. epidermidis, increases the rate of opsonization and phagocytosis of a variety of Staphylococcus infections, and induces enhanced intracellular killing of Staphylococcus bacteria.
It is another object of the present invention to provide an immunological serum against staphylococci.
It is another object of the present invention to provide such a serum which yields humoral and cellular immunity against staphylococci.
It is another object of the present invention to provide such a serum which imparts short-term immunity against staphylococci.
It is a further object of the present invention to provide methods for detecting, diagnosing, treating, preventing or monitoring the progress of therapy for staphylococcal infections.
A method and composition for the passive immunization of patients infected with or susceptible to infection from Staphylococcus bacteria such as S. aureus and S. epidermidis infection is provided that includes the selection or preparation of a donor plasma pool with high antibody titers to carefully selected Staphylococcus adhesins or MSCRAMMs, or fragments or components thereof, or sequences with substantial homology thereto; purification, concentration, and treatment of the donor plasma pool as necessary to obtain immunoglobulin in a purified state that has a higher than normal antibody titer to the selected adhesins; and then administration of an effective amount of the purified immunoglobulin to the patient in need thereof. The donor plasma pool can be prepared, for example, by combining individual blood or blood component samples which have higher than normal titers of antibodies to one or more of the selected adhesins or other proteins that bind to extracellular matrix proteins, or fragments or sequences with substantial homology thereto, to produce the desired composite. Kits for the identification of donor plasma pools with high titers of the selected adhesins are also provided. In an alternative embodiment, a method for obtaining a donor plasma pool that is highly effective against Staphylococcus bacterial infection is provided that includes administering carefully selected proteins or peptides to a host to induce the expression of desired antibodies, recovering the enhanced high titer serum or plasma pool from the host, optionally purifying and concentrating the immunoglobulin, and providing it to a patient in need thereof.
A xe2x80x9chigh titerxe2x80x9d of antibody in this context means the presence of an antibody which is immunoreactive with the selected adhesin or fragment thereof which is 2-fold or greater, e.g., up to 10-20 more times higher than that found in a normal population of 100 random samples of blood or blood components.
In one embodiment of the invention, a donor plasma composition is selected or prepared that has a high titer of antibodies to at least a fibrinogen binding protein, such as Clumping factor A (xe2x80x9cClfAxe2x80x9d) or Clumping factor B (xe2x80x9cClfBxe2x80x9d), or fragments or components thereof, or a protein or fragment with sufficiently high homology thereto.
In another embodiment of the invention, a donor plasma composition is selected or prepared that has a high titer of antibodies to at least a collagen binding protein or peptide (or an appropriate site directed mutated sequence thereof), a fragment or component thereof, such as the collagen binding domain protein M55, or a protein or fragment with sufficiently high homology thereto.
In another embodiment of the invention, a donor plasma composition is selected or prepared that has a high titer of antibodies to at least a fibronectin binding protein or peptide (or an appropriate site directed mutated sequence thereof), or a protein or fragment with sufficiently high homology thereto, as well as the fibrinogen binding protein A and B (ClfA or ClfB), or useful fragments or components thereof or a protein or fragment with sufficiently high homology thereto.
In a further embodiment, a donor pool is selected or prepared that has a high titer of antibodies to at least the fibrinogen binding protein A (ClfA) and the fibrinogen binding protein B (ClfB), or useful fragments thereof or a protein or fragment with sufficiently high homology thereto.
In a still further embodiment, a donor pool is selected or prepared with a high titer of antibodies to at least a fibronectin binding protein or peptide (or an appropriate site directed mutated sequence thereof), or a protein or fragment with sufficiently high homology thereto, in combination with (I) high titer antibodies to the fibrinogen binding protein A and B (ClfA and ClfB), or a useful fragment thereof or a protein or fragment with sufficiently high homology thereto; and (ii) high titer antibodies to a collagen binding protein or useful fragment thereof.
In another embodiment, a donor pool is selected or prepared that has a high titer of antibodies as in any of the previous embodiments in combination with a high titer of antibodies to an elastin binding protein or peptide or a protein or fragment with sufficiently high homology thereto.
In another embodiment, a donor pool is selected or prepared that has a high titer of antibodies as set forth in the embodiments above in combination with high titers of antibodies to a MHC II analogous protein or peptide or a protein or fragment with sufficiently high homology thereto.
In an additional embodiment, a donor pool is selected or prepared that has a high titer of antibodies to any of the embodiments above in combination with high titer of antibodies to one or more fibrinogen binding proteins SdrC, SdrD or SdrE, or useful fragments thereof or proteins or fragments with sufficiently high homology thereto.
In still another embodiment, a donor pool is selected or prepared that has a high titer of antibodies to at least the fibrinogen binding protein SdrC, the fibrinogen binding protein SdrD and the fibrinogen binding protein SdrE or useful fragments thereof or a protein or fragment with sufficiently high homology thereto.
Kits are also provided that identify plasma pools with high titers of the desired antibodies. In one embodiment, a suitable amount of antibodies to antibodies of the combination of proteins or peptides as described herein can be immobilized on a solid support and are preferably labeled with a detectable agent. Antibodies can be immobilized to a variety of solid substrates by known methods. Suitable solid support substrates include materials having a membrane or coating supported by or attached to sticks, beads, cups, flat packs, or other solid support. Other solid substrates include cell culture plates, ELISA plates, tubes, and polymeric membranes. The antibodies can be labeled with a detectable agent such as a fluorochrome, a radioactive label, biotin, or another enzyme, such as horseradish peroxidase, alkaline phosphatase and 2-galactosidase. If the detectable agent is an enzyme, a means for detecting the detectable agent can be supplied with the kit. A preferred means for detecting a detectable agent employs an enzyme as a detectable agent and an enzyme substrate that changes color upon contact with the enzyme. The kit can also contain a means to evaluate the product of the assay, for example, a color chart, or numerical reference chart.
Preferably, the isolated immunoglobulin is of the IgG fraction or isotype, but isolated immunoglobulin is not restricted to any particular fraction or isotype and may be IgG, IgM, IgA, IgD, IgE, or any combination thereof. It is also preferable that the isolated immunoglobulin be purely or antigenically human immunoglobulin, which may be obtained from human sources or made directly by the fusion of human antibody producing cells with human antibody producing cells or by the substitution of human DNA sequences for some of the nonhuman DNA sequences which code for the antibody while retaining the antigen binding ability of the original antibody molecule.